1. Molecular Biology & Biotechnology
Mohammad Tahir Sm
M.Sc 4 th Semester
Molecular Biology & Biotechnology

3. Isolation of Different Strains of Genus Streptomyces
for the Production of Antibiotics

4. Introduction
Streptomycetes are the important source of antibiotics in medical, veterinary and agriculture field.
Gram positive
Non motile
Spore forming
Fungal like growth
Antibiotic Production

5. Actinomycetaceae family and specifically to the Streptomyces genus, the members of which have antimicrobial activity.
It has been estimated that the genus Streptomyces might produce at least 1,00,000 new compounds of biological interest.

6. Streptomycin is an antibiotic produced by the soil microbe Streptomyces griseus.
Selman Waksman isolated the microbe in 1943, and reported its antimicrobial activities in 1944.
This was the first antimicrobial agent developed after penicillin.

7. They belong to the class of bacteria which have very important role for human welfare and are known as actinomycetes.
Cohn was the first scientist who performed his work on the Streptomyces species and the name Streptomyces was then conferred by Selman Walksman in 1943.

8. Antibiotics may be classified as bactericidal
&
bacteriostatic.
Streptomycin is an antibiotic produced by the soil microbe Streptomyces griseus.

9. Mode of Action
Streptomycin is bactericidal in action. It inhibits protein synthesis by combining irreversibly with the 30S subunit of the70S ribosomes, found typically in prokaryotes. Specifically, it binds with the S12 protein involved in the initiation of protein synthesis.
Streptyomycin is also known to prevent the normal dissociation of 70S ribosomes into their 50S and 30S subunits. Thus formation of polysomes is inhibited.
The overall effect of streptomycin seems to involve distorting the ribosome so that transition from initiation complex (30S-mRNA-tRNA) to chain elongating ribosome is blocked.
Thus, the normal sequence of translation is disrupted, the bacteria is unable to synthesize proteins vital for its cell growth and thereby fails to survive. The drug also disrupts the cell membrane of susceptible bacteria.

10. Objectives
To screen the soil of province Punjab to explore the diversity of genus Sreptomyces and to screen commercially important members of this genes.
To evaluate the production of antibiotics of these isolates using standard strains of the bacteria.

11. Materials and Methods:
Soil sampling
The soil sampling will be carried out from the agricultural land of the province Punjab. 30 soil samples will be collected 8 inch deep and will be taken in sterilized zipper plastic bags.The samples will be stored at 4˚C for further use.

12. Soil Enrichment
One gram of soil will be taken in a crucible dish and heated in an oven at 45˚C for 12 hours.
The samples will be mixed with 0.1 g calcium carbonate and incubated at 26˚C for 7 days in water bath.

13. Growth Media
Chemically defined media will be prepared using following composition (g/l). The pH will be adjusted at 6.90 and the medium will be autoclaves at 121°C for 15 min.
Na2HPO4 1
NaH2PO4 1
MgSO4 1
CaCl
FeSO
K2HPO4 1
Glycerol 0.5

14. Antimicrobial assay
After 96 hours culture broth will be centrifuged at 5000 rpm for 10 minutes to separate cells.
The cell free broth will be analyzed for the presence of antibiotic.
Nutrient agar plates will be prepared and holes will be made in the solidified agar plates.
Then cell free broth will be added into the hole.
Bacterial lawn will be prepared in which Staph aureus, Escherichia coli and Bacillus subtilis will be used as a test organism.
The antimicrobial activity of streptomyces will be observed through formation of zone of inhibition against the test organism.

15. Analysis of Antibiotics
Antibiotics production will be analyzed by the thin layer chrometographic technique (TLC).
Standard antibiotics will be compared with each bacterial metabolites.